The epidermis is faced with the daunting task of generating a permeability barrier that allows survival of mammals in a xeric, terrestrial environment. To accomplish this goal, the epidermis generates the stratum corneum (SC), with its highly-hydrophobic, lipid-enriched extracellular matrix, organized into lamellar bilayers. To generate these unique protective structures, keratinocytes must synthesize extensive quantities of cholesterol and sphingolipids, along with free fatty acids (FA), and deliver an approximately equimolar ratio of these barrier lipids to the SC. These three key lipids, and/or their immediate precursors, are removed from the intracellular, membrane-lipid pool by sequestration within an epidermis-specific, secretory organelle, the lamellar body, and delivered to the SC interstices coincident with terminal differentiation. Coordinate regulation of the synthesis of these lipid classes, and their subsequent sequestration into lamellar bodies, not only sustains a flow of lipids in an appropriate equimolar ratio to meet permeability barrier requirements, but also quickly funnels newly-synthesized ceramides (Cer) into glucosylceramides (GIcCer), thereby avoiding premature, Cer-induced apoptosis. It is our hypothesis that epidermal lipid synthesis is co-regulated and co-ordinated with both cholesterol and FA production: e.g., as cholesterol and FA synthesis increase in response to barrier requirements, nuclear hormone receptor-activating oxysterol and free fatty acid metabolites will be generated, which in turn up-regulate Cer and GIcCer synthesis. Conversely, when cholesterol and FA synthesis declines, fewer lipid metabolites will be generated and NHR-dependent activation of SPT and GCS also will decline. We propose first, to identify those NHR agonists that regulate Cer and GIcCer synthesis in relation to differentiation, and then to determine the basis, as well as the receptor-mediated requirement for such regulation (Aim 1). After identifying the most-potent NHR activators, we will determine how SPT and GCS are regulated by these agents at a molecular level (Aim 2). Finally, we will assess how Cer/GIcCer production are co-regulated with cholesterol and FA synthesis, to generate the equimolar mixture required for the barrier (Aim 3). Since the formation of the epidermal barrier lipids, and subsequent generation of the extracellular lamellae, are late steps in epidermal differentiation, the proposed studies will result in significant new information regarding the coordinate regulation of barrier lipid generation and epidermal homeostasis.